Method of determining odorant compounds and antagonists of odorants using a primary culture of olfactory neurons

ABSTRACT

Primary cultures of purified olfactory neurons can be stimulated with physiological levels of odorants. The neurons of the cultures express markers characteristic of mature olfactory neurons in vivo, such as vimentin, olfactory marker protein and neuron-specific enolase. The cultures are useful for screening for odorants and antagonists, as well as for biochemical and physiological studies of olfactory transduction. The olfactory neurons may comprise at least about 85% of cells in the culture. The olfactory neurons demonstrate responsiveness in culture to IBMP, citraliva, and isovaleric acid.

This invention was made with government support under grants DA-00266and NS-01231 awarded by The United States Public Health Service. Thegovernment has certain rights in the invention.

This application is continuation of Ser. No. 07/769,210, filed Oct. 1,1991, now abandoned, which is a divisional of Ser. No. 07/633,513, filedDec. 31, 1990, now U.S. Pat. No. 5,217,893.

TECHNICAL FIELD OF THE INVENTION

This invention relates to the area of primary cell culture. Morespecifically it relates to culture of olfactory neuronal cells.

BACKGROUND OF THE INVENTION

Olfactory transduction discriminates with great accuracy and sensitivityamong a multitude of volatile, low molecular weight compounds (Lancet,D. (1986) Ann. Rev. Neurosci., 9:329-355; Snyder, S. H., Sklar, P. B.and Pevsner, J. (1988) J. Biol. Chem., 263:13971-13974), but compared tovisual transduction, it is relatively poorly understood. One difficultylies in the heterogeneity of olfactory epithelium in which the olfactoryneurons reside. The olfactory primary sensory neurons are located in apseudostratified columnar epithelium consisting of three principal celltypes (Graziadei, P.P.C. (1971) The olfactory mucosa of vertebrates. In:Handbook of Sensory Physiology, Vol. I. (Ed. Beidler, L. M.),Springer-Verlag, Berlin, pp. 27-58). The sustentacular or supportivecells resemble glial cells and stretch from the epithelial surface ofthe basal lamina. Cell bodies of the sensory neurons lie at variouslevels in the epithelial layer and extend apical dendrites to thesurface of the epithelium and unmyelinated axons through the basallamina. The third cell type, the basal cell, underlies the receptorneurons and is thought to serve as a precursor population from which newolfactory neurons can arise. Isolation of receptor neurons from theseother cell types has been difficult, thereby limiting ability to performbiochemical analysis.

Several attempts have been made to obtain populations of primaryolfactory neurons. Initial efforts employed in vitro culture of theentire olfactory epithelium (Gonzales, et al., (1985) J. Neurosei.Methods, 14:77-90; Noble et al., (1984) Neurosei. Letts, 45:193-198).N-ethylmaleimide has been used to dissociate olfactory epithelium cellsinto single cells, which, however, lose excitable properties (Kleene, S.J. and Gesteland, R. C. (1981) Brain Res., 229:536-540). Hirsch, J. D.and Margolis, F. L. (1979) Brain Res., 161:277-291, have employedenzymatic dissociation followed by general mechanical disruption withdissociated cells centrifuged through a bovine serum albumin (BSA)gradient, yielding a partially purified population of cells. Others(Calof, A. L. and Chikaraishi, D. M., (1989) Neuron, 3:115-127; Pixley,S. K. and Pun, R. Y. K. (1990) Develop. Brain Res., 53:125-130) havedevised methods to perform lineage analysis and electrophysiologicstudies on embryonic olfactory neuronal cells but the neuronal cellswere not in pure cultures and were therefore not amenable to biochemicalstudies.

Thus there is a need in the art for relatively pure populations ofprimary olfactory neurons which retain their excitability in response toodorants.

BACKGROUND OF THE INVENTION

It is an object of the invention to provide a primary culture ofolfactory neurons, substantially purified from other olfactoryepithelial cells.

It is another object of the invention to provide a primary culture ofolfactory neurons which is responsive to odorants.

It is yet another object of the invention to provide a method ofproducing primary cultures of olfactory neurons.

It is still another object of the invention to provide a test kit fordetermining the effects of odorants on olfactory neurons.

It is an object of the invention to provide a method of identifyingodorants.

It is still another object of the invention to provide a method foridentifying antagonists of known odorants.

These and other objects of the invention are provided by one or more ofthe embodiments which are described below. In one embodiment of theinvention a primary culture of olfactory neurons is provided which issubstantially purified from sustentacular cells and basal cells. Thecultured neurons demonstrate responsiveness to physiologic levels ofodorants. The cultured neurons express vimentin, olfactory markerprotein and neuron-specific enolase. The cultured neurons do not expressglial fibrillary acidic protein, S-100 protein, keratin, orneurofilament protein.

In another embodiment of the invention a method of producing primarycultures of olfactory neurons is provided. The method comprises thesteps of:

providing olfactory epithelium of an animal;

disrupting the olfactory epithelium to separate cells of said olfactoryepithelium;

passing the separated cells of said olfactory epithelium through a meshhaving a pore size of between about 10 and 25 microns to remove cellaggregates;

plating said separated cells in a nutrient medium comprising D-valine,cytosine arabinoside, and nerve growth factor (NGF), on a solidsubstrate.

In still another embodiment of the invention a test kit is provided fordetermining the effects of odorants on olfactory neurons. The test kitcomprises: a primary culture of olfactory neurons, substantiallypurified from sustentacular cells and basal cells on a solid support,said neurons demonstrating responsiveness to physiologic levels ofodorants, said neurons expressing vimentin, olfactory marker protein andneuron-specific enolase, said neurons not expressing glial fibrillaryacidic protein, S-100 protein, keratin, or neurofilament protein; and ameans for testing neuronal excitation.

In yet another embodiment of the present invention a method foridentifying antagonists of known odorants is provided. The methodcomprises the steps of:

exposing the primary culture of olfactory neurons of the presentinvention to a known odorant and determining a first level of excitationof the neurons;

exposing the primary culture of olfactory neurons to a mixture of theknown odorant and a putative antagonist and determining a second levelof excitation of the neurons;

comparing said first and second excitation levels, an antagonist beingidentified when the second level of excitation is less than the firstlevel of excitation.

In still another embodiment of the invention a method for identifyingnew odorants is provided. The method comprises the steps of:

exposing the primary culture of olfactory neurons of the presentinvention to a putative odorant and determining a level of excitation ofthe neurons, an odorant being identified which increases the level ofexcitation of the culture over a basal level, said basal leveldetermined in the absence of the putative odorant.

These and other embodiments of the invention which will be described inmore detail below, provide the art with a potent tool to investigateolfactory transduction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows phase-contrast microscopy of cultures of olfactory neurons.FIG. 1A is within 2 hours of plating; FIG. 1B is 24 hours after plating;FIG. 1C is 72 hours after plating; FIG. 1D shows a low-magnificationimage at 72 hours. FIG. 1E shows neuronal processes stained withanti-vimentin antibodies and fluorescenated secondary antibody.

FIG. 2 shows immunocytochemistry of neonatal rat nasopharyngealsections. FIG. 2A, low-power of coronal section stained for OMP. FIG.2B, a more caudal section demonstrates OMP-positivity in the epitheliumand on fibers converging on the bulb. FIG. 2C, low-power coronalsections stained for vimentin. FIG. 2D, staining of olfactory epitheliumfor OMP. FIG. 2E, staining for vimentin. FIG. 2F, staining of olfactoryepithelium for neuron specific enolase. FIG. 2G, staining of olfactorytissue for tubulin. FIG. 2H, staining of olfactory epithelium withanti-keratin antibodies. FIG. 2I, staining of olfactory epithelium forGFAP. FIG. 2J, staining with non-immune control serum.

FIG. 3 shows immunocytochemical staining of primary cultures of ratolfactory neurons. Phase-contrast (FIGS. 3A, 3C, 3E, 3G, 3I, and 3K) andimmunofluorescent (FIGS. 3B, 3D, 3F, 3H, 3J, and 3L) images are shown.FIGS. 3A and 3B, olfactory neuronal culture stained positively for OMP.FIGS. 3C and 3D, anti-vimentin antibodies stain olfactory neuronsdiffusely, highlighting neurites. FIGS. 3E and 3F, there is nodetectable staining with anti-S-100 antibodies. FIGS. 3G and 3H neuronspecific enolase antibodies stain olfactory cell bodies. FIGS. 3I and3J, occasionally, a GFAP positive cell can be visualized which hasextremely different morphology. FIGS. 3K and 3L, non-immune serumreveals no detectable staining.

FIG. 4 shows a Western blot analysis of extracts of olfactory neurons inculture. Tissue from olfactory bulb (b), olfactory epithelium (E), orprimary cultures of olfactory neurons (C) were prepared. Extracts wererun on PAGE and Western blot analysis performed. All three extracts showdetectable OMP (lanes 1-3). Likewise, vimentin is present in all threetissues (lanes 4-6). GFAP is detectable in olfactory bulb andepithelium, but not in cultures (lanes 7-9). S-100 is detectable inbulb, but not in epithelium or cultured material (lanes 10-12). Both NSE(lanes 13-15) and tubulin (lanes 16-18) are detectable in all threetissues. No staining is seen when non-immune serum is utilized (lanes19-21).

FIG. 5 demonstrates immunocytochemical staining of cells trapped byfiltration during preparation of primary cultures. Primary cultures wereprepared. Cellular material trapped during filtration was resuspended inmedium and plated. Phase-contrast (FIGS. 5A, 5C, 5E, 5G, and 5I) andimmunofluorescent image (FIGS. 5B, 5D, 5F, 5H, 5J) pictures are shown.FIGS. 3A and 3B, epithelial cell clusters do not stain positively forvimentin; however, occasionally a trapped putative olfactory neuronalcell is seen, which does stain positively for vimentin. FIGS. 3C and 3D,epithelial clusters do not demonstrate positive staining for OMP. FIGS.3E and 3F, anti-keratin antibody recognizes small clusters of cells,which may represent aggregates of basal cells. FIGS. 3G and 3H, athigher magnification, the large cluster of putative epithelial cellsdoes not stain for keratin, while the small cluster seen is quitepositive. FIGS. I and J, non-immune serum demonstrates no staining inthese cultures.

FIG. 6 demonstrates an evaluation of various matrix substrate substancesfor morphology and plating efficiency of olfactory neurons. Cellcultures were prepared and plated on substrates. Phase-contrast (FIGS.6A, 6C, 6E, 6G, 6I, 6K, 6M, 6O, 6Q, and 6T) and phase-contrastimmunofluorescent (FIGS. 6B, 6D, 6F, 6H, 6J, 6L, 6N, 6P, 6R, and 6T)images are shown. FIGS. 6A and 6B, laminin; FIGS. 6C and 6D,fibronectin; FIGS. 6E and 6F, fibronectin and laminin; FIGS. 6G and 6H,poly-D-lysine; FIGS. 6I and 6J, polyornithine (PORN); FIGS. 6K and 6L,laminin and PORN; FIGS. 6M and 6N, heparan sulfate; FIGS. 6O and 6P,collagen; FIGS. 6Q and 6R, collagen IV; FIGS. 6S and 6T, collagen IV andlaminin treatment.

FIG. 7 shows the effect plating of primary cultures onto matrigelcompared to laminin. FIG. 7A, laminin; FIG. 7B, matrigel; FIG. 7Cmatrigel at higher magnification.

FIG. 8 demonstrates the effect of NGF on olfactory neuron survival.Olfactory neurons were grown either with (FIG. 8A) or without (FIG. 8B)NGF at a concentration of 25 ng/ml.

DETAILED DESCRIPTION OF THE INVENTION

It is the discovery of the present invention that primary cultures ofolfactory neuronal cells can be obtained which retain theirresponsiveness to odorants in vitro. These cultures are substantiallypurified from other cell types, including other olfactory epithelialcells, fibroblasts and mesenchymal cells. The high level of purity ofthe cells which has been obtained renders the cultures highly suitablefor biochemical studies of olfactory transduction. The polyclonality ofthe cell cultures suggests that they encompass the entire spectrum ofodorant responsiveness.

The cultures of the present invention contain predominantly olfactoryneurons. Typically they contain greater than about 85% neurons. Oftenthey contain greater than about 90% neurons. Preferably they containgreater than about 95% or 98% neurons. The level of purity can beassessed by a number of criteria. Certain proteins are characteristic ofolfactory neuronal cells and their presence can be determined andquantitated. These include olfactory marker protein (OMP),neuron-specific enolase (NSE), and vimentin. Other proteins arecharacteristically absent from olfactory neuronal cells and theirpresence can also be determined and quantitated. These include S-100protein, glial fibrillary acidic protein, neurofilament protein, andkeratin. As is known in the art there are many ways to determine thepresence of particular proteins in cell populations. Any of these may beused in the assessment of purity of the cell cultures. Particularlysuitable methods are immunological, and include immunohistochemistry andWestern blotting.

The cells of the olfactory epithelium from which the neuronal cells havebeen purified are mainly the sustentacular cells and the basal cells.These cells can be distinguished on the basis of morphology, markerproteins, and lack of excitability. The cultured neuronal cells of thepresent invention are responsive to physiologic levels of odorants,which may vary from odorant to odorant. However, generally these areexceedingly low levels, in the range of 1 nM to 10 uM. For example, theneuronal cultures of the present invention are stimulated to 50% oftheir maximal adenylyl cyclase augmentation at 1 nM by2-isobutyl-3-methoxypyrazine (IBMP), whereas comparable levels ofexcitation are elicited by between about 1-10 uM of citralva orisovaleric acid.

Primary cultures comprise cells which are taken directly from a wholeanimal source. They are not continuous cultures, and have a limitedtime-span of survival in culture. Typically the life-span in culture isin the range of 1 to 2 weeks. The neuronal cells of the presentinvention are typically mature cells and do not divide.

The neuronal cell cultures of the present invention may be derived fromany of a variety of animal sources. For example, chickens, rats, andfrogs have been used to study olfaction previously and are suitable foruse in the present invention. Preferred animal sources are mammals.Although the applicants do not wish to be bound by any particulartheory, neonatal tissue may be advantageous as a source material for theneuronal cell cultures.

According to the method of the present invention, the olfactoryepithelium cells are obtained by dissection as is known in the art. Asuspension of olfactory epithelium cells is prepared by means ofmechanical and enzymatic disruption of the tissue structure. The tissuecan first be minced to provide small pieces of tissue, e.g., ofapproximately 1 mm². A combination of enzymes may be used to separatethe cells from each other, including hyaluronidase, DNAse, collagenase,and dispase. Typically these enzymes are incubated with the tissue at37° C. to digest the substances which may bind and aggregate the cellsto each other. The tissue can also be mechanically disrupted by anymeans known in the art, such as trituration, Waring blender,Potter-Elvehjem homogenizer, meat grinders, shakers, etc.

According to the method of the present invention, neuronal cells arepurified from other olfactory epithelium cells by means of a sizefractionation procedure. The size fractionation can be convenientlyaccomplished by passing a suspension of epithelial cells through a meshof defined pore size or spacing intervals. The meshes may be made of anymaterials although wire and nylon meshes may be convenient and readilyavailable. The meshes may range in size from about 150 microns to 10microns, preferably from about 25 to 10 microns. Preferably theepithelial cell suspensions are passed through meshes of increasinglysmaller sizes. The size fractionation purifies neuronal cells becausethe other cells of the olfactory epithelium aggregate in larger clumpsthan the mesh and thus can be filtered out from the cell suspensions.The neuronal cells appear to dissociate into single cell units and notto re-aggregate, therefore permitting passage through the pores of themeshes.

The size fractionated cells can be resuspended in a nutrient medium.Many nutrient media are known in the art for animal cells and may beused in the practice of the present invention. However, it has beenfound to be particularly advantageous to include D-valine, and cytosinearabinoside in the medium as selective agents. D-valine is selective forepithelial cells, and inhibits growth of fibroblasts and mesenchymecells which may contaminate the neuronal cell suspension. Cytosinearabinoside inhibits the growth of dividing cells, thereby selectingmature, non-dividing cells, such as the neuronal cells. It is a findingof the present invention that NGF (nerve growth factor) which isnecessary for growth for some types of neurons but not others, isnecessary for survival of olfactory neurons.

The cell suspensions of the present invention can be plated on a solidsupport such as a glass slide or plastic tissue culture dish. It ispreferred that a matrix substance such as laminin be adhered to thesolid support to provide a solid substrate for attachment of the cells.Laminin provides an additional degree of selectivity to the method ofculturing neuronal cells, because neuronal cells attach to it, butfibroblasts do not. Other matrix substances such as fibronectin orMatrigel™ (reconstituted basement membrane, available from CollaborativeResearch) can also be used, although laminin appears to provide superiorresults.

The present invention also contemplates test kits for determining theeffects of odorants on olfactory neurons. The kits comprise the primarycultures of olfactory neurons of the present invention on a solidsupport. In addition, a means for testing neuronal excitation isincluded. The means for testing neuronal excitation can be thesubstrates for measuring cyclic AMP, which is produced upon excitationof neurons. Typically the means for testing cyclic AMP levels will bethe components of a radioimmunoassay, such as is commercially availablefrom NEN/DuPont, Boston, Mass. Other means for testing for neuronalexcitation can also be employed, such as patch clamp recordingtechniques.

The neuronal cell cultures of the present invention can be used toscreen compounds for their effects on olfactory tissues. For examplecompounds can be screened for their ability to stimulate the neuronalcells above their basal level of excitation. Such compounds are known asodorants. Stimulation can be measured by any means known in the art tocorrelate with excitation of the neuronal cells. These means includeelectrophysiological measurements such as patch clamp recording, as wellas biochemical means such as measuring the accumulation of cyclic AMP orthe increase in specific activity of adenylyl cyclase.

Alternatively, compounds can be screened for their ability to block theexcitation of neuronal cells by known odorants. Such compounds arecalled antagonists. Antagonism can readily be determined by exposing thecultures of the present invention to the odorant alone, and to theodorant in admixture with a putative antagonist. A decrease in the levelof excitation in the presence of the putative antagonist relative to thecontrol levels in the presence of odorant only, identifies anantagonist.

EXAMPLE 1

This example describes the isolation of purified olfactory neuronal cellcultures.

Olfactory tissue from neonatal rats was collected, minced, dissociatedin enzymes and plated on chamber slides pretreated with laminin asdescribed below.

Approximately 3 litters or 28-32 pups were used per prep. The 2-3 dayold rat pups were sacrificed by decapitation, and olfactory tissuedissected and immediately placed in modified Eagle's medium (MEM)containing 4.8 g/l of HEPES buffer, designated MEM-AIR. The turbinateswere transferred twice through fresh MEM-AIR to minimize contamination.Tissue was then centrifuged at 700×g for 7 min. After the supernatantwas decanted, the tissue was minced to achieve tissue fragments ofapproximately 1 mm in size, resuspended in MEM-AIR and centrifuged at700×g for 7 min. Tissue was then placed in 30 ml of MEM-AIR containing1% (w/v) BSA, RIA grade (Sigma, St. Louis, Mo.), 1 mg/ml hyaluronidase(Sigma), 50 ug/ml DNAse (Sigma), 1 mg/ml collagenase (WorthingtonBiochemicals, Freehold, N.J.), and 5 mg/ml dispase (Boehringer-MannheimBiochemicals, Indianapolis, Ind.), and incubated with agitation for 1 hrat 37° C. At the end of incubation, the cell suspension was triturated10 times with a 10 ml plastic piper and passed through a 150 micron wiremesh. The cell suspension was then centrifuged at 500×g for 5 min. Thesupernatant was aspirated and the cell pellet resuspended in platingmedium composed of modified Eagle's medium containing D-valine (MDV,Gibco, Grand Island, N.Y.) containing 15% (v/v) dialyzed fetal calfserum (dFCS, Gibco), 5% (v/v) NU serum (Collaborative Research, Bedford,Mass.), 10 uM cytosine arabinoside (ara C) and 25 ng/ml nerve growthfactor (NGF, Collaborative Research). After resuspension, cells weresuccessively filtered through 50 micron and 10 micron nylon mesh filters(Small Parts, Miami, Fla.), to remove any remaining undigested clumps ofcells and epithelial cells. Cells were plated at a density of 1×10⁶cells per cm² into tissue culture dish (Falcon, Lincoln Park, N.J.) orLabtek tissue culture slides (Nunc, Naperville, Ill.) coated with MDVcontaining laminin at 25 ug/ml (Collaborative Research). Cultures wereplaced in a humidified 37° C. incubator receiving 5% CO₂. On day 2 andevery day thereafter, cells were fed with MDV containing 15% dFCS,gentamicin, kanamycin, NGF and ara C.

Within 2 hr after plating, cells attach and began to spread (FIG. 1A).After 24 hr in culture, cells began to distribute more uniformly on theculture surface and extended a short multi-branched process and anopposing long unbranched process (FIG. 1B). At 72 hr in culture, cellsreached a uniform distribution (FIG. 1C), and most non-neuronal cellsdied off. Neurite outgrowth appears random (FIG. 1D); cell processes areextremely long, about 20-50 cell body lengths and can be bettervisualized by immunofluorescence, using anti-vimentin antibodies (FIG.1E).

EXAMPLE 2

This example describes immunocytochemical and immunoblottingcharacterization of the primary cultures of the invention.

Immunohistochemistry was performed on neonatal rats the same age asthose utilized in primary culture preparation. Animals were perfusedwith PBS and then 4% paraformaldehyde in PBS. Olfactory tissue wasdissected and post-fixed in 4% PFA for 2 hr at room temperature.Thereafter, tissue was sunk in PBS containing 15% (w/v) sucrose,embedded in brain paste and sectioned on a cryostat.

For immunocytochemistry, cells were plated in two-chamber Labtek tissueculture slides previously treated with MDV containing 25 ug/ml laminin.After 5-7 days in culture, the upper chamber of the slides was removedand slides were rinsed quickly three times at 37° C. inphosphate-buffered saline (PBS), pH 7.3. Slides were immediately placedin PBS containing 4% (w/v) paraformaldehyde at 37 ° C. and incubated for20 min. Alternatively for immunofluorescence, primary culture cellsplated on slides were quickly rinsed 3 times with PBS at 37° C., andplaced in methanol at -20° C. for 15 min. Monolayers treated in eithermanner were then washed 3 times for 5 min each in PBS at roomtemperature, permeabilized by incubation in 0.1% (w/v) Triton-X-100(T-X-100) for 15 min, and rinsed again 3 times in PBS. When chromagenwas used, endogenous peroxidase activity was quenched by incubation ofslides in PBS containing 2% (v/v) hydrogen peroxide, followed by 3rinses in PBS. Nonspecific staining was blocked by incubation for 1 hrwith non-immune serum, appropriate for the secondary antibody, at adilution of 1:100 in PBS containing 1% (w/v) BSA. Slides were thenincubated in PBS containing 1% BSA and primary antiserum overnight at 4°C. The next day, the slides were rinsed three times with PBS, blockedwith PBS containing 1% BSA and incubated for 2 hr at 25° C. with theappropriate biotinylated secondary antibody using Vectastain kits(Vector Labs, Burlingame, Calif.). Slides were again washed in PBS,blocked, incubated for 1 hr in PBS containing 1% BSA in theavidin-biotin-horseradish peroxidase complex (Vector Labs), rinsed, andincubated for 5 min with the chromogen, 3-amino-9-ethylcarbazole (AEC,Biomeda Corporation, Foster City, Calif.). For immunofluorescence,primary antiserum at the appropriate dilution was added to slides in PBScontaining 1% BSA and incubated overnight at 4° C. The next day, slideswere washed three times for 5 min each in PBS and incubated in PBScontaining 1% BSA and the appropriate fluorescenated or rhodaminatedantibody (Jackson Immunoresearch Laboratories Incorporated, West Grove,Pa). Slides were subsequently washed three times in PBS, and phase andfluorescent pictures were immediately taken.

Antisera were used at the following dilutions: monclonal anti-vimentinantibody (Boehringer-Mannheim) at 1:3 dilution; polyclonal anti-neuronspecific enolase (NSE) antibody (IncStar, Stillwater, Minn.) at 1:4dilution; polyclonal anti-S-100 antibody (IncStar) at 1:4 dilution;polyclonal anti-tubulin antibody (Dr. Douglas Murphy, Johns HopkinsUniversity School of Medicine, Baltimore, Md.) at 1:250 dilution;polyclonal anti-olfactory marker protein (OMP) antibody (gift of Dr.Frank Margolis, Roche Institute, Nutley, N.J.) at 1:300 dilution;anti-glial fibrillary acidic protein (GFAP, DAKO-Patts, Santa Barbara,Calif.) at 1:800 dilution; and non-immune serum as control (Vector Labs)at 1:100 dilution.

At low magnification (FIGS. 2A-C), the olfactory epithelium appears asfrond-like projections extending towards the midline septum. Theolfactory epithelium and the primary axons, which extend towards thebulb, stain positively for OMP (FIGS. 2A, B). Unlike other neuronalcells, the axons of olfactory neurons contain the intermediate filamentvimentin (Schwob et al., 1986 J. Neurosci. 6:208-217), whoseimmunoreactivity is apparent in olfactory primary axons as they convergeupon the olfactory bulbs (FIG. 2C). At higher magnification, while OMPis evident in the epithelium containing neuronal cell bodies and thesubmucosal axonal process (FIG. 2D), vimentin is localized in thesubmucosal region containing the primary axons (FIG. 2E). Neuronspecific enolase (NSE) immunoreactivity is also localized selectively toolfactory mucosa (FIG. 2F). Tubulin occurs diffusely throughout themucosa, but with greater density at the luminal surface, which containscilia (FIG. 2G). Anti-keratin antibodies stain the basal cell layer(FIG. 2H). Anti-GFAP antibodies stain the submucosal region (FIG. 2I),presumably reflecting glial-like cells which surround olfactory axons.Staining of olfactory tissue with non-immune serum is shown in FIG. 2J.

Like olfactory neurons in intact tissue, olfactory neurons in primaryculture stain positively using anti-OMP antibodies (FIGS. 3A, B),anti-vimentin antibodies (FIGS. 3C, D), and anti-NSE antibodies (FIGS.3G, H). No staining of primary cultures is seen using anti-S-100antibody (FIGS. 3E, F). Occasional GFAP-positive cells are seen (FIGS.3I, J), which have distinctive morphology; none of the bipolar cellsstain positively for GFAP. No staining is seen when non-immune serum isused in place of primary antiserum (FIGS. 3K, L). Thus, cultures of ratprimary olfactory neurons stain specifically for those markers found inneurons in vivo. Immunofluorescence was used for primary cultures aspositive staining in the processes is not adequately demonstrated byperoxidase.

To confirm the specificity of staining in both tissue sections and inprimary cultures of olfactory neurons, Western blot analysis wasperformed (FIG. 4).

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) wasperformed according to the method of Laemmli (1971), utilizing a 14-20%gradient. Western blotting employed the method of Speicher (1980) Proc.Natl. Acad. Sci. USA, 77:5673-77, with several modifications. Gels weretransferred to nitrocellulose paper at 60 V for 5 hrs at 4° C. At theend of this time, gels were rinsed in water and blocked in buffercontaining 0.15M NaCl, 0.05M Tris-HCl, 0.05% sodium azide, and 2% (w/v)RIA-grade BSA for 12 hr at 4° C. The next day, blots were washed 3 timesin PBS. For rabbit antisera, the primary antiserum was placed inblocking buffer containing antiserum at the appropriate dilution, 2% BSAand 0.05% tween-20. Incubation was performed overnight at 4° C. The nextday, blots were washed three times in PBS containing 0-0.05% tween.Blots were incubated in ¹²⁵ I-protein A in blocking buffer containing 2%BSA and 0.05% tween for 1 hr at room temperature. Thereafter, blots werewashed 3 times in PBS with 0.05% tween, and exposed to X-omat (Kodak,Rochester, N.Y.) film. For monoclonal mouse antibodies, blots werewashed in PBS without tween and incubated in primary antibody at theappropriate dilution in blocking buffer containing 2% BSA and 3% normalserum overnight at 4° C. The next day, blots were rinsed 5 times withPBS, and incubated in blocking buffer containing 2% BSA and rabbitanti-mouse IgG at 1:100 dilution (Jackson Labs) for 1 hr at roomtemperature. Blots were again rinsed 3 times in PBS and mouse peroxidaseanti-antiperoxidase complex (Sternberger-Meyer, Jarrettsville, Md.) at1:200 dilution in blocking buffer with 2% BSA for 1 hr at roomtemperature. Blots were again rinsed 3 times in PBS, once in 50 mM Tris,pH 7.6 and then incubated in AEC for 30 sec.

Tissue was collected from olfactory bulb (B), olfactory epithelium (E)and primary olfactory cultures (C). Extract (100 ug per lane) was loadedon a 14-20% SDS-PAGE gel, electrophoresed, and transferred tonitrocellulose paper. Blots were incubated with antibodies against OMP(FIG. 4, lanes 1-3), anti-vimentin antibodies (FIG. 4, lanes 4-6),anti-GFAP antibodies (FIG. 4, lanes 7-9), anti-S-100 antibodies (FIG. 4,lanes 10-12) anti-NSE antibodies (FIG. 4, lanes 13-15), anti-tubulinantibodies (FIG. 4, lanes 16-18) and non-immune serum in place ofprimary antiserum (FIG. 4, lanes 19-21). Antibodies against OMPrecognize a band of appropriate molecular weight in all three tissues.Anti-vimentin antibodies recognize a band of appropriate molecularweight in bulb an olfactory epithelium extract, as would be expectedgiven the mesenchymal elements present in these tissues, as well as inthe primary olfactory cells whose axons contain vimentin. Althoughantibodies against GFAP detect a band of appropriate mobility inolfactory bulb and olfactory epithelium, no such band is seen uponincubation with olfactory cultured extracts. While immunofluorescence(FIG. 3I and J) demonstrates the presence of occasional GFAP-positivecells, levels of GFAP are presumably too low to be detected by Westernblotting. Antibodies against S-100 protein recognize a band ofappropriate molecular weight in olfactory bulb, but not in olfactoryepithelium or primary cultures. NSE immunoreactivity of appropriatemolecular weight is evident in bulb, olfactory epithelium and primarycultures. Antibodies against tubulin detect a doublet of appropriatemolecular weight in all three tissues, while non-immune serum fails toidentify the major band sin any of the three extracted tissues. Thus,Western blot analysis confirms the specificity of staining seen byimmunocytochemistry of primary cultures of rat olfactory neurons.

EXAMPLE 3

This example describes the immunocytochemical characterization of cellsremoved from the primary cell cultures by filtration according to themethod of the invention.

A procedure to prepare olfactory neurons from dissociated cells ofolfactory mucosa includes entrapment of epithelial cells on a 50 micronfilter just prior to plating. Perhaps due to differences inextracellular matrix components, the epithelial cells, which probablyinclude epithelial and sustentacular cells, remain associated orreassociate so that they are trapped by filtration. Some neurons aretrapped as well. Epithelial cells generally appear flat and polygonal,but after 5-7 days in cultures they may appear fusiform, although theydo not extend processes similar in length or diameter to the primaryneurons seen in the flow-through. The epithelial cells trapped on thefilters were characterized immunocytochemically (FIG. 5). Cellularmaterial trapped on filters was eluted into plating medium and plated ina similar manner to olfactory neurons. Fixation and immunocytochemicalstaining was performed as described above.

In contrast to olfactory neurons, epithelial cells do not stain forvimentin (FIGS. 5A and B). An occasional putative neuronal cell isretained along with the epithelial cells and does stain for vimentin(FIGS. 5A and B). Epithelial cells do not stain for OMP (FIGS. 5C andD). Basal cells are apparently trapped on filters as well, as smallclumps of cells stain positively for keratin found in basal cells invivo FIGS. 5E-H). No staining is seen using non-immune serum (FIGS. Iand J). Thus, a substantial part of cellular selection is achieved byfiltration, which apparently traps the majority of epithelial cells.Although after a week in culture these cells acquire spindle-shapedmorphology, they are initially rounded and polygonal and therefore caneasily be differentiated from neurons. In addition, their stainingpatterns differ from neuronal cells.

EXAMPLE 4

This example describes the effects of various substrate matrixsubstances on culture characteristics of olfactory epithelium cells.

To determine the role of substrate matrix substances on platingefficiency and on cellular selection, a number of different substrateswere tested.

Two chamber slides were pretreated in a number of ways. For laminincoating, 1 ml of MDV containing 25 ug/ml laminin was plated onto glasstwo-chamber slides overnight at 37° C.; before use, the slides wererinsed with MDV. For fibronectin, slides were treated with a solutioncontaining 1 ml of MDV containing 20 ug/ml fibronectin (CollaborativeResearch), incubated overnight at 37° C., and rinsed with MDV prior touse. For polyornithine (PORN, Sigma Chemicals), a solution at aconcentration of 1 mg/ml PORN in deionized distilled water was added toslides, incubated 1 hr at 37° C., rinsed twice with deionized distilledwater, and rinsed once with MDV prior to use. When laminin was used onPORN-treated slides, slides were treated with PORN and then laminin inthe usual manner. Coating of slides with poly-D-lysine was performedanalogous to treatment with polyornithine. Heparan sulfate was dilutedin MDV to a concentration of 10 ug/ml and 1 ml was placed on a slide,maintained overnight at 37° C., and rinsed with deionized distilledwater prior to use. Collagen (Gibco) or collagen IV (CollaborativeResearch) was prepared as a 50 ug/ml solution in deionized distilledwater and 1 ml of this solution utilized per slide. In the case ofcollagen, the solution was allowed to air dry. Collagen IV wasmaintained overnight at room temperature, aspirated, air dried and thenrinsed twice with deionized distilled water prior to use. When lamininwas used with collagen IV, collagen IV was prepared as described, andlaminin was subsequently added in the usual manner. Matrigel(Collaborative Research) was utilized according to manufacturer'sinstructions.

Primary cultures were prepared as describe above and plated onto anumber of different substrates and examined by phase contrast as well asimmunofluorescence with antivimentin antisera (FIG. 6). Cells maintainedon laminin (FIG. 6A and B) resemble those grown on fibronectin (6D andE), though the processes are shorter with the latter substrate.Combining laminin and fibronectin (FIGS. 6E and F) provides anappearance similar to laminin alone. Cells maintained on poly-D-lysine(FIGS. 6G and H) display poor plating efficiency and minimal processoutgrowth, while with polyonrithine (FIGS. 6I and J) they appear onlyslightly better than with poly-D-lysine. Laminin and polyornithinetogether (FIGS. 6K and L) afford better plating efficiency and neuriteoutgrowth, which remains inferior to laminin or fibronectin.Heparan-sulfate (FIGS. 6M and N) yields similar results topolyornithine. Although collagen (FIGS. 6O and P) allows somewhat betterplating efficiency than poly-D-lysine, polyornithine or heparan-sulfate,process outgrowth is still stunted and plating efficiency is notoptimal. Collagen IV (FIGS. 6Q and R) yields improved neurite outgrowthand plating efficiency, which is still inferior to fibronectin orlaminin. Utilization of laminin and collagen IV together (FIGS. 6S andT) yields better plating efficiency and neurite outgrowth, which isinferior to laminin or fibronectin. Thus, of all substrates tested,laminin consistently provides optimal plating efficiency and therefore,has been used for subsequent experimentation.

Matrigel was also examined as a substrate (FIG. 7). On laminin, cellsextend neurites in a random fashion, with no tendency to reaggregate(FIG. 7A). By contrast, when duplicate suspensions of cells are platedonto matrigel, the cells aggregate over the next 48 hr to form clusters(FIGS. 7B and C). Some cells extend perpendicularly from these clusters,with the extending process demonstrating multiple short, branchedprocesses. Thus, in a three dimensional matrix, these culture cellsassume a polarity, with structures representing putative cilia extendingapically.

EXAMPLE 5

This example describes the role of NGF in maintaining olfactory neuronalcells in culture.

NGF Is thought to play an important role in neuronal maturation anddifferentiation (for review, see Levi et al., (1988) Misko, Radeke etal., (1987) J. Exp. Biol., 132:177-190. To investigate the role of NGFon olfactory neuronal survival and neurite extension, primary cultureswere plated in the absence or presence of NGF at concentrations of 25ng/ml (FIG. 8). Primary cultures were plated into two-chamber slides. Analiquot of medium with (FIG. 8A) or without (FIG. 8B) NGF was addedafter cells were plated. Although both sets of cells initially extendprocesses with good plating efficiency, by 3 days in culture those cellsmaintained without NGF lose process extension and cell viability isdecreased by approximately 85%. Therefore, NGF apparently affects botholfactory neuronal survival and neurite extension in culture.

EXAMPLE 6

This example describes the excitation of primary olfactory neuroncultures by odorants.

After 5-7 days in primary culture, olfactory neuronal cells were exposedto odorants and cyclic AMP levels assayed. To avoid transient changes inintracellular cyclic AMP levels caused by feeding, the medium waschanged to 0.5 ml of MEM-AIR containing 0.1% (w/v) RIA-grade BSA 2-3 hrbefore use. At zero-time, cultures received an additional 0.5 ml ofMEM-AIR with or without 10 uM 2-isobutyl-3-methoxypyrazine (IBMP). Atvarious times thereafter the entire medium was aspirated and thereaction quenched by addition of 6% in cold TCA. Cellular extracts wereprocessed and RIA for cyclic AMP was performed as described below.

Primary cultures of olfactory neurons were plated in laminin-treated24-well cluster dishes at a density of 1×10⁶ cells per cm². Cells wereused between days 5-7 in culture. Thirty rain before experimentation,monolayers were fed with MEM-AIR containing 0.1% (w/v) RIA grade BSA.Immediately prior to use, odorants were diluted from a 50 mM stock inabsolute ethanol to 10 uM. At zero time, 0.5 ml of MEM-AIR containing0.1% RIA grade BSA and odorant at twice the final concentration wasadded to wells.

The experiment was terminated by aspiration of culture medium andaddition of 200 ul of 6% (w/v) trichloroacetic acid (TCA) at 0° C.Culture wells were heated to 56° C. in an oven for 1 hr, scraped,sonicated, chilled, and microfuged for 30 see. The supernatants wereextracted 4 times with 5 volumes of ether, and the ether-extracted waterphase was lyophilized to dryness. Samples were resuspended in 200 ul ofRIA-NEN buffer (NEN/DuPont, Boston, Mass.) and RIA assay was performedaccording to specifications of the cyclic AMP kit.

                  TABLE I                                                         ______________________________________                                        Effect of Odorant on cAMP Levels in Culture Cells                             time (min)       cAMP (% of control ± SEM)                                 ______________________________________                                        Primary olfactory neurons                                                     0.5              350 ± 160                                                 1.0              250 ± 92                                                  5.0              270 ± 113                                                 15               175 ± 47                                                  NG-108 cells                                                                  0.5              103 ± 19                                                  1.0              94 ± 32                                                   5.0              99 ± 16                                                   15               70 ± 41                                                   ______________________________________                                    

IBMP elicits a rapid, transient rise in intracellular cyclic AMP levels(Table 1). The maximum increase seen is approximately three-fold overcontrol levels. Similar treatment of NG-108 cells does not alter cyclicAMP levels.¹

We claim:
 1. A method for identifying antagonists of known odorants,comprising the steps of:contacting a primary culture of dissociatedolfactory neurons with a quantity of a known odorant, said quantity ofknown odorant having the ability to excite said olfactory neurons, saidolfactory neurons:(a) comprising at least about 85% of the cells in saidprimary culture of olfactory neurons, (b) demonstrating responsivenessin culture to 1 nM IBMP, to between about 1 and about 10 uM citralva,and to between 1 and about 10 uM isovaleric acid, (c) expressingvimentin, olfactory marker protein and neuron-specific enolase, and (d)not expressing glial fibrillary acidic protein, S-100 protein, keratin,or neurofilament protein, said expression being assessed byimmunohistochemistry or by Western blotting; determining a first levelof excitation of the neurons in the presence of said quantity of theknown odorant; contacting said primary culture of olfactory neurons witha mixture consisting of said quantity of the known odorant and aputative antagonist; determining a second level of excitation of theneurons in the presence of said mixture; and comparing said first andsecond excitation levels, an antagonist being identified when the secondlevel of excitation is less than the first level of excitation.
 2. Amethod for identifying compounds as odorants which were not previouslyknown to be odorants, comprising the steps of:contacting a primaryculture of dissociated olfactory neurons with a compound to be tested,said olfactory neurons:(a) comprising at least about 85% of the cells insaid primary culture of olfactory neurons, (b) demonstratingresponsiveness in culture to 1 nM IBMP, to between about 1 and about 10uM citralva, and to between 1 and about 10 uM isovaleric acid, (c)expressing vimentin, olfactory marker protein and neuron-specificenolase, and (d) not expressing glial fibrillary acidic protein, S-100protein, keratin, or neurofilament protein, said expression beingassessed by immunohistochemistry or by Western blotting; determining alevel of excitation of said olfactory neurons in the absence of thecompound; determining a level of excitation of said olfactory neurons inthe presence of the compound; comparing the determined levels ofexcitation of the neurons in the presence and absence of the compound,the compound being identified as an odorant which increases the level ofexcitation of said olfactory neurons.
 3. The method of claim 1 whereinsaid olfactory neurons comprise at least about 90% of the cells in saidprimary culture of olfactory neurons.
 4. The method of claim 2 whereinsaid olfactory neurons comprise at least about 90% of the cells in saidprimary culture of olfactory neurons.
 5. The method of claim 1 whereinsaid olfactory neurons comprise at least about 95% of the cells in saidprimary culture of olfactory neurons.
 6. The method of claim 2 whereinsaid olfactory neurons comprise at least about 95% of the cells in saidprimary culture of olfactory neurons.
 7. The method of claim 1 whereinsaid olfactory neurons comprise at least about 98% of the cells in saidprimary culture of olfactory neurons.
 8. The method of claim 2 whereinsaid olfactory neurons comprise at least about 98% of the cells in saidprimary culture of olfactory neurons.